Vascular Damage, Thromboinflammation, Plasmablast Activation, T-Cell Dysregulation and Pathological Histiocytic Response in Pulmonary Draining Lymph Nodes of COVID-19

This study aims to characterize pathophysiological changes in lethal COVID-19 lymph nodes. 22 lethal COVID-19 cases and 28 controls were enrolled in this study. Pulmonary draining lymph nodes (mediastinal, tracheal, peribronchial) were collected at autopsy. Control lymph nodes were selected from a range of histomorphological sequelae [unremarkable histology, infectious mononucleosis, follicular hyperplasia, non-SARS related HLH, extrafollicular plasmablast activation, non-SARS related diffuse alveolar damage (DAD), pneumonia]. Gene expression profiling was performed using the HTG EdgeSeq Immune Response Panel. Characteristic patterns of a dysregulated immune response were detected: 1. An accumulation of extrafollicular plasmablasts with a relative paucity or depletion of germinal centers. 2. Evidence of T-cell dysregulation demonstrated by immunohistochemical paucity of FOXP3+, T-Bet+ and LEF1+ positive T-cells and a downregulation of key genes responsible for T-cell crosstalk, maturation and migration as well as a reactivation of herpes viruses in 6/21 COVID-19 lymph nodes (EBV, HSV). 3. Macrophage activation by a proinflammatory, CD163+ phenotype and increased incidence of hemophagocytic activity. 4. Microvascular dysfunction, evidenced by an upregulation of hemostatic (CD36, PROCR, VWF) and proangiogenic (FLT1, TEK) genes and an increase of fibrin microthrombi and CD105+ microvessels. Taken together, these findings imply widespread dysregulation of both innate and adoptive pathways with concordant microvascular dysfunction in severe COVID-19. Overall design: Examination of autopsy lymph node tissue of lethal COVID-19 cases versus controls by gene expression profiling Please note that the 'VLP00581_AI_Plate1_09JAN2021_Parsed-forReveal_QualityControlled.xlsx' processed data file contains 78 data columns, while 50 samples (used for the final manuscript of the study) are included in the records. The extra data columns refer to either universal RNA samples used for correlations tests by HTG Molecular, which are labelled uRNA on the second sheet 'QC_Raw' of the count matrix excel file, -or- samples which failed the internal quality controls of HTG Molecular, such as poor quality of sample (Q0), insufficient read depth (Q1) or expression variability (Q2), marked on the third sheet 'QC_summary'. Further samples were excluded as described in the forthcoming manuscript, such as inadequate patient data.

本研究旨在明确致死性新型冠状病毒肺炎（COVID-19）患者淋巴结的病理生理变化。本研究共纳入22例致死性COVID-19病例与28例对照样本。于尸检阶段采集肺引流淋巴结，包括纵隔淋巴结、气管旁淋巴结、支气管周围淋巴结。对照淋巴结选自多种组织形态学后遗症：组织学无异常、传染性单核细胞增多症、滤泡增生、非SARS相关噬血细胞性淋巴组织细胞增生症（HLH）、滤泡外浆母细胞活化、非SARS相关弥漫性肺泡损伤（DAD）、肺炎。本研究采用HTG EdgeSeq免疫反应检测板（HTG EdgeSeq Immune Response Panel）进行基因表达谱分析。

研究检测到免疫应答失调的特征性模式：1. 滤泡外浆母细胞蓄积，同时生发中心相对匮乏或耗竭；2. T细胞失调的证据：免疫组化检测显示FOXP3+、T-Bet+及LEF1+阳性T细胞数量减少，且参与T细胞串扰、成熟与迁移的关键基因表达下调；此外21例COVID-19淋巴结中有6例检出疱疹病毒活化，即EB病毒（EBV）与单纯疱疹病毒（HSV）；3. 巨噬细胞呈促炎表型（CD163+）并活化，且噬血细胞活性发生率升高；4. 微血管功能异常，表现为止血相关基因（CD36、PROCR、血管性血友病因子（VWF））及促血管生成基因（FLT1、TEK）表达上调，同时纤维蛋白微血栓与CD105+微血管数量增加。综上，上述发现提示重症COVID-19患者的先天免疫与适应性免疫通路均存在广泛失调，且伴随微血管功能异常。

整体实验设计：通过基因表达谱分析，对比致死性COVID-19病例与对照的尸检淋巴结组织。

请注意，处理后的数据集文件'VLP00581_AI_Plate1_09JAN2021_Parsed-forReveal_QualityControlled.xlsx'包含78个数据列，而本研究最终手稿所用的50份样本已纳入记录。额外的数据列要么对应HTG Molecular用于相关性检验的通用RNA样本（在计数矩阵Excel文件的第二张工作表“QC_Raw”中标记为uRNA），要么对应未通过HTG Molecular内部质量控制的样本，例如样本质量不佳（Q0）、测序读长不足（Q1）或表达变异度异常（Q2），此类样本在第三张工作表“QC_summary”中已标注。另有部分样本已如后续发表手稿所述被排除，例如患者临床数据不全。